This invention relates to a method for transferring DNA sequences, including a description of the unique DNA sequences, capable of transferring non-host resistance between plant species.
Through evolution only a relatively small group of microorganisms have found their pathological niche on a given plant species. Fortunately, the same niche is not usually available for this same microbe group in a different plant species. Thus, the "different" plant species is said to express "non-host" resistance (Heath, M. C. Non-host resistance in: Plant Disease Control; Resistance and Susceptibility. R. C. Staples and G. H. Toehniessen, Eds. John Wiley and Sons, Inc., New York, p. 201, 1981.) to this group of microorganisms. This mismatch is often manifest as an intense host plant response (hypersensitive response) based on the activation of a selection of host genes (Daniels, C. H., Fristensky, B. W., Wagoner, W. W. and Hadwiger, L. A. Pea genes associated with non-host disease resistance to Fusarium are also active in race-specific disease resistance to Pseudomonas. Plant Molecular Biology, 8, 309, 1986; Fristensky, B. W., Riggleman, R. C., Wagoner, W. W. and Hadwiger, L. A. Gene expression in susceptible and disease resistant interactions of peas induced with Fusarium solani pathogens and chitosan. Physiol. Plant Pathol., 27, 15, 1986.). Non-host resistance has recently attained a new importance with the advent of genetic engineering, because it is now possible to transfer genes from a species which actively resists a given plant pathogen to a species which is infected by that pathogen (Hadwiger, L A., Chiang, C. C., Pettinger, A., and Chang, M. M. Strategy to improve disease resistance by transferring "non-host" disease resistance genes from peas to potatoes. University of Maryland--USDA--DuPont, 2nd Int'l. Symp. on Biotechnology and Food Safety, Butterworth, Boston, 1990.). Theoretically, there is a limitless source of potential disease resistance genes existing in all of the plant species on earth, this invention describes a method for transferring specific DNA sequences from a pea (Pisum sativum) plant to transformable plant species such as tobacco and potato. it defines the state of knowledge of how these genes are controlled and defines a combination of genes needed to code for that portion of the intense host response which actually suppresses the pathogen and preserves the viability of host cells (Kendra, D. F. and Hadwiger, L A. Cell death and membrane leakage not associated with the induction of disease resistance in peas by chitosan or Fusarium solani f. sp. phaseoli, Phytopathol., 77, 100, 1987.) adjacent to the point of pathogen challenge. Conventional plant breeding techniques have used pathogen race-specific genes (Fehr, W. R. Principals of cultivar development. V. 1, Ch.21, Breeding for pest resistance. MacMillan Pub. Co., N.Y., 304-13. 1987.) available within a given plant species to improve commercial crop species. This disease resistance which evolved through reassortment of traits via intra-specific crossing and subsequent natural selection, "Single Mendelian traits" (McIntosh, R. A. A catalog of gene symbols for wheat [1983 edition], Proc. 6th Int'l. Wheat Genet. Symp. Kyoto, Japan, 1983, 1197, 1983.) can provide the plant with race-specific resistance to races evolving within a given microbe species. Race-specific resistance is often attributable to a match-up of a single dominant trait in the host with a single dominant trait in the pathogen which is called a gene-foregene interaction (Flor, H. H. Current status of the gene-foregene concept. Annu. Rev. Phytopath., 9, 275, 1971.).
In addition to non-host resistance and race-specific resistance there is the phenomena of induced resistance. Plants which have no genetically definable traits for disease resistance can often generate disease resistance by induced resistance. Induced resistance occurs when a plant, prechallenged by a non-virulent microbe, is subsequently inoculated with a virulent pathogen (Kuc', J. and Preisig, C. Fungal regulation of disease resistance mechanisms in plants, Mycologia, 76, 767, 1984.). As a result, the virulent pathogen is often resisted. Another amazing aspect of induced resistance is the observation that a challenge by a compatible virulent pathogen can also induce a resistance response which retards infection by subsequent inoculation of the same pathogen in adjacent leaves. Induced resistance can also occur following the application of a compound capable of eliciting a disease resistance response (Hadwiger, L A. and Beckman, J. M. Chitosan as a component of pea-F. solani interactions. Plant Physiol., 66, 2305, 1980). Often disease resistance does not function properly if metabolic inhibitors (Heath, M. C. Effects of heat shock, actinomycin D, cycloheximide, and blasticidin S on non-host interactions with rust fungi. Physiol. Plant Pathol., 15, 211, 1979.) heat shock, etc. (Chamberlain, D. W. and Gerdemann, J. W. Heat-induced susceptibility of soybeans to Phytophtophthora megasperma var. sojae, Phytophthora cactorum, and Hebninthosporium sativum., Phytopathology, 5670, 1966.) are utilized to interfere with the development of a resistance response (Hadwiger, L. A. and Wagoner, W. W. Effect of heat shock on the mRNA-directed discase resistance response of peas, Plant Physiol., 72, 553, 1983.). Such observations clearly demonstrate that a plant has the biochemical capability to resist nearly any pathogen if its response is generated both quickly and completely enough to ward off the pathogen.
A definition of non-host resistance in terms of classical genetics, e.g. is not presently possible, because it is seldom possible to make inter-species crosses and follow the chromosomal inheritance of resistance traits within a progeny. However, it is possible to follow genes whose MRNA and protein products accumulate in abundance as non-host resistance is being expressed, Additionally, there is evidence which suggests this specific intense defense response is necessary for disease resistance (Hadwiger, L. A., Wagoner, W. W. Electrophoretic patterns of pea and Fusarium solani proteins synthesized in vitro which characterize the compatible and incompatible interactions. Physiol. Plant Pathology 153, 1983.). My laboratory has defined major genes in the pea/Fusarium solani f. sp. phaseoli interaction which distinguish the resistance reaction from the susceptible reaction between pea and Fusarium solani f. sp. isi. The cloned response genes of the non-host resistance response and their regulators (promoters) have been transferred to potato plants and been shown to respond quickly and intensely to plant pathogenic stains normally infecting their species.
1. Field of the Invention
The field of the invention is primarily that of genetic improvements of plants through genetic engineering. Ibis invention centers on the use of unique DNA sequences to improve the ability of crop plants to resist diseases.
2. Description of the Prior Art
Conventional plant breeding has been the major and most successful method of improving the disease resistance of crop plants. Genetic traits controlling disease resistance have been identified in plants by genetic crosses which reveal both the distribution of a functional gene (master gene) in gene types and often its location on a chromosome. The first successful engineering of a disease resistance gene occurred when Dr. Ernest Sears of the University of Missouri was able to desegment a chromosomal fragment of a grassy plant and re-associate it with a normal wheat chromosome in an inter-specific cross. The re-associated chromosome piece transferred to wheat a major genetic trait for resistance to a rust-inciting organism. It was realized early that such translocation of chromosome segments often result in the co-transfer of detrimental traits. More recently genetic engineering technology has enabled the transfer of individual genes both within and between plant species.
To date because of the great abundance of repetitive DNA sequences in the region of the plant chromosome which containing the major Mendelian genes controlling discase resistance, attempts to define these major gene DNA sequences have been unsuccessful.
This has generated a second level effort to define genes which become active in a disease resistance response. Some of these genes code for functional proteins such as the enzymes which carry out secondary metabolic pathways in plants (Bailey). There is an abundance of DNA sequence information in the literature for these genes which code for enzymes such as phenylalanine ammonia lyase, cinnamic acid hydrolase, chalcone synthetase and peroxidases (Dixon review). Also, DNA sequences have been obtained for plant structural compounds which increase somewhat after pathogen challenge, for example the hydrolyproline rich proteins. DNA sequences are also available for plant genes which produce hydrolytic enzymes which are capable of degrading individual cellular components of the invading pathogenic organisms. Genes for enzymes such as .beta.-glucanase which degrades .beta.-glucan and chitinase which digests fungal wall chitin have been sequenced and researched extensively. Some or all of these genes probably contribute to the plant's defense. My laboratory has shown that the hydrolytic enzymese .beta.-glucanase and chitinase are capable of releasing from fungal cell walls a small oligomer of chitosan which is capable of suppressing the growth of both virulent and avirulent pathogens for a limited period. However, these compounds appear not to be ultimately determinant in developing complete disease resistance as are the genetic components coding of the enzymes of secondary plant pathways. The major entity of complete disease resistance is cell vitality, in the cells which encircle the point of challenge by the pathogenic organism. In resistance reactions only a few cells in contact with the pathogen lose vitality, whereas in a susceptible reaction a broad radius of cells lose vitality. Paramount to the multiplicity of resistance response associated with plant defense is the speed of the resistance response. Vitality enhancing products must accumulate in cells prior to the appearance of the pathogenic propagule which is releasing its destructive components. The genomic DNA sequences in this invention contain promoters which we have established act rapidly when present inherently in the donor pea. These promoters also act equally well to enhance the output of their associated structural gene products, or when fused with foreign structural genes, enhance other gene products when genetically transferred to a recipient plant species. We have also determined that the product of Gene 49 accumulates in the nuclei of plant cells in the radius of cells which remain viable in a resistance reaction, in an apparent cause and effect relationship.